EP0484374A1 - Rapid, versatile and simple system for expressing genes in eukaryotic cells. - Google Patents
Rapid, versatile and simple system for expressing genes in eukaryotic cells.Info
- Publication number
- EP0484374A1 EP0484374A1 EP90910982A EP90910982A EP0484374A1 EP 0484374 A1 EP0484374 A1 EP 0484374A1 EP 90910982 A EP90910982 A EP 90910982A EP 90910982 A EP90910982 A EP 90910982A EP 0484374 A1 EP0484374 A1 EP 0484374A1
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- European Patent Office
- Prior art keywords
- protein
- rna
- cell
- virus
- cat
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12N9/1033—Chloramphenicol O-acetyltransferase (2.3.1.28)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
- C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32211—Cardiovirus, e.g. encephalomyocarditis virus
- C12N2770/32241—Use of virus, viral particle or viral elements as a vector
- C12N2770/32243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- An object of the present invention is to provide an efficient expression system which confers cap-independent translation to RNAs in eukaryotic cells, particularly when a prokaryotic transcription system is used in eukaryotic cells.
- An additional object of the present invention is to provide a method for controlled expression of toxic genes.
- Figure 1 shows a schematic structure of expression cassettes in accordance with the present invention.
- P ⁇ 7 bacteriophage T7 010 promoter from -23 to +1, 5'hp, nucleotides +1 to +26 downstream of the 010 promoter and capable of forming a hairpin or stem-loop structure
- T ⁇ 7 bacteriophage T7 010 termination sequence
- EMC nucleotides 163 to 746 of the EMCV TJTR.
- FIG. 2 shows the results of time course study of CAT synthesis during vaccinia virus infection.
- CV-l cell monolayers were infected with 20 PFU per cell of wild-type vaccinia virus (lane 9) , or co-infected with VTF7-3 and VT7CAT (lanes l, 2, 5, 6, 10, 11), or with VTF7-3 and VT7EMCAT (lanes 3, 4, 7, 8, 12, 13) at a multiplicity of 10 PFU per cell of each virus.
- VTF7-3 and VT7CAT las 2, 5, 6, 10, 11
- VTF7-3 and VT7EMCAT las 3, 4, 7, 8, 12, 13
- Figure 3 demonstrates the accumulation of CAT protein by 24 hr after infection.
- CV-l cell monolayers were infected with 20 PFU per cell of wild-type vaccinia virus (A, lane l; B, solid bar) , or co-
- VTF7-3 and VT7CAT A, lane 2; B, hatched bar
- VTF7-3 and VT7EMCAT a, lanes 3 and 4; B, open bar
- the isotonic medium was maintained (A, lanes 1-3; B, part 1) or changed to 190 irtM (hypertonic) NaCl (A, lane 4;#B part 2) .
- cell lysates were prepared, 20 ⁇ g of protein were loaded in each lane of a 10% NaDodS ⁇ 4 -polyacrylamide gel and following electrophoresis the gel was stained with Coomassie brilliant blue (A) or CAT activity was determined as described in the text.
- Figure 4 shows cap-independent translation of RNA containing EMCV UTR.
- In vitro transcription was carried out using Bam HI digested pT7CAT (lanes 1-4) or pT7EMCAT (lanes 5-8) as templates. Lanes containing unmethylated (GpppG) or methylated (m 7 GpppG) capped RNA are indicated.
- Translation in micrococcal-treated rabbit reticulocyte lysate was performed with RNA concentration of 8 nM with or without the presence of 0.1 mM methylated GDP (m 7 GDP) as indicated.
- Figure 5 demonstrates polyribosome-association and in-vitro translation of CAT RNA made in infected cells.
- HeLa cells were co-infected with 10 PFU/cell of VTF7-3 and 10 PFU/cell of either VT7CAT
- RNA polymerase and promoter are from a DNA bacteriophage and the ribosome binding site is from an RNA virus.
- the preferred embodiment comprises a recombinant vaccinia virus constructed to include a T7 promoter, an encephalomyocarditis virus (ECMV) untranslated region (UTR) , and a T7 promoter regulated gene for a protein desired to be expressed, as illustrated in Figure 1.
- ECMV encephalomyocarditis virus
- UTR encephalomyocarditis virus
- the untranslated region that confers cap independent translation may be from sources other than EMCV including related viruses such as poliovirus and mengovirus and from unrelated viruses such as adenovirus and the like.
- sources other than EMCV including related viruses such as poliovirus and mengovirus and from unrelated viruses such as adenovirus and the like.
- SUBSTITUTESHEET prokaryotic transcription systems such as bacteriophage SP6, T3 and GH1 could be employed for expression in eukaryotic cells.
- CV-l monkey kidney cells were propa ⁇ at_?r_ -. «? mrmniavers in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were infected as described previously (Fuerst et al, 1987, Mol. Cell Biol. 7, 2538-2544; Fuerst et al, 1989, J. Mol. Biol. 206:333-348). Plasmid constructions. A 0.6 kilobasepair (kbp) EcoRl- Ball EMC fragment from pE5LVPO (Parks et al, 1986, J. Virol.
- nucleotides 163 to 746 of the EMCV UTR was inserted into the blunted BamHl site of plasmid vector pAR2529 (Rosenberg et al, 1987, Gene 56, 125-135 to generate pTF7.25EMC-l.
- the CAT coding sequence was isolated from pTF7CAT-l (Fuerst et al, supra), as 0.8 kbp Taq 1 fragment and using BamHl linkers was inserted into the BamHl site of pUC18 to generate the plasmid pUC18CAT-l.
- the 0.8 kbp BamHl CAT fragment was then isolated from pUC18CAT-l and inserted into a unique BamHl site of plasmid pTF7.25EMC-l downstream of the major translation start site of EMC, to form plasmid pTF7.25EMCAT-l.
- the coding frame of CAT was placed immediately downstream of EMC start codon by site directed mutagenesis. The sequence immediately- flanking the initiating EMC ATG was changed to 5'- EMC...GATAATACCATGGAG...CAT-3' , which provides a unique Ncol site.
- SUBSTITUTESHEET referred to as pT7EMCAT and pT7CAT, respectively.
- a deposit of pT7EMCAT plasmid has been made at the ATCC, Rockville, Maryland on July 7, 1989 under the accession number 68045.
- the deposit shall be viably maintained, replacing if it becomes non-viable during the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and upon issuance of the patent made available to the public without restriction in accordance with the provisions of the law.
- the Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
- Vaccinia virus insertion vectors were prepared by excising the complete T7 promoter-target gene-T7 terminator cassettes as Bglll fragments, blunting the ends and inserting into the blunted EcoRl site of pGS50 (Fuerst et al, supra). This resulted in flanking the target gene cassettes with vaccinia virus thymidine kinase (TK) sequences used for homologous recombination. Recombination into the genome of vaccinia virus and the isolation of recombinant virus was carried out as described by Mackett et al, 1984, J. Virol. 49:857-864. In vitro transcription.
- TK thymidine kinase
- RNA in the translation reaction mixture were 8 nM or 80 ng/ ⁇ l, respectively.
- CAT assay was measured as described by Shaw (1975, Methods in Enzymolocrv 43, 737-748), with modifications. Confluent CV-l cells in a 6-wells plate were coinfected and grown with minimum essential medium without Phenol Red (Quality Biological, Inc.) and supplemented with 2.5% fetal bovine serum. At 24 hr after infection, the cells and medium were collected and lysis was achieved by addition of NaDodS04 and chloroform to 0.0005% and 1%, respectively. Samples
- isolation and purification techniques are employed. Such techniques include centrifugation, filtration, chromatography, electrophoresis, dialysis and the like well known to one of ordinary skill in the art.
- CAT activity was measured 24 hr after transfection of each T7 promoter plasmid into cells that had been infected with the helper virus VTF7-3 (Fuerst et al, supra) , which express the T7 RNA polymerase.
- VTF7-3 helper virus
- An approximately 7-fold enhancement provided by the EMCV UTR was reproduced in several independent experiments.
- CAT level expressed by a T7EMCAT plasmid which did not contain the T7 stem-loop was 10 fold less than T7EMCAT indicating that the T7 stem-loop is necessary for expression despite the fact that EMCV UTR is expected to have considerable secondary structure.
- Both pT7CAT and pT7EMCAT contain vaccinia virus thymidine kinase sequences flanking the expression cassette making it simple to generate recombinant vaccinia viruses VT7CAT and VT7EMCAT.
- CAT expression was measured after infecting cells simultaneously with either T7 promoter-containing recombinant virus and VTF7-3 (to provide T7 RNA polymerase) .
- the overall results were similar to those obtained by transfection with the corresponding plasmids except that the F.MCV TJ ⁇ P en anced expression 4- to 5-fold in repeated experiments.
- CV-l cells were infected with VTF7-3 to provide T7 RNA polymerase and either VT7CAT or
- VT7EMCAT Protein synthesis was monitored by NaDodS0 4 - polyacrylamide gel electrophoresis of cytoplasmic proteins from cells labeled with [ 35 S] ethionine at various times after infection in isotonic or hypertonic (190 mM) medium ( Figure 2) . At 2 hr after infection, predominantly host proteins were labeled under all conditions (lanes 1-4) . However, by 6 hr. host protein synthesis was inhibited and the late pattern of protein synthesis was established (lanes 5-8) .
- the CAT protein was the predominant band expressed by VT7EMCAT under isotonic conditions (lane 12) and was even more dominant under hypertonic conditions (lane 13) . Densitometer tracings of the fluorogram indicate that at.24 hr. 78% of the [ 35 S]methionine was
- the total accumulation of CAT protein at 24 hr after infection was determined by Coomassie brilliant blue staining of NaDodS0 4 -polyacrylamide gels as well as by assaying CAT activity ( Figure 3) .
- Cells were co- infected in isotonic medium and then either maintained under these conditions or were transferred to hypertonic medium after 4 hr. After a total of 24 hr, the cells were lysed and 20 ⁇ g of cytoplasmic protein were loaded on each lane of a 10% polyacrylamide gel.
- RNAs with and without m 7 G capped 5'-ends were synthesized RNAs with and without m 7 G capped 5'-ends and compared their in vitro translation properties.
- Bacteriophage T7 RNA polymerase was used to transcribe the plasmids pT7CAT and pT7EMCAT (Figure 1) in order to prepare mRNAs with and without ⁇ _-._ « FM ⁇ . UTR.
- Transcripts containing 5' methylated caps were generated by adding high concentrations of m 7 GpppG to the transcription reaction; GpppG was added to parallel reactions to generate unmethylated caps which are known to be non-functional in translation but may also protect the RNAs against exonuclease degradation. These transcripts were translated in a rabbit reticulocyte lysate in the presence or absence of the cap analog m 7 GDP, known to be a specific inhibitor of methylated capped RNA translation in vitro. In the absence of the EMCV UTR, translation of T7-CAT transcripts was found to be methylated cap-dependent as the GpppG-terminated RNA was inefficiently translated relative to that of m 7 GpppG-terminated RNA ( Figure 4) .
- the polyribosome-associated and the free CAT RNA were pooled separately and translated in rabbit reticulocyte lysates in the presence or absence of m7GDP. Inspection of fluorograms of NaDodS0- polyacrylamide gels ( Figure 5B) led to several significant findings. First, both the polyribosome- bound and free T7-EMCV UTR-CAT RNA was translated more efficiently than either T7-CAT RNA fraction. Second, translation of the polyribosome-bound T7-CAT RNA was inhibited by m 7 GDP whereas the corresponding free RNA was not inhibited. Third, translation of neither the polyribosome nor the free T7 EMCV UTR-CAT RNA was inhibited by m 7 GDP.
- T7-CAT transcripts were selectively associated with the polyribosome fraction accounting for the inhibition of their translation by m 7 GDP.
- the free T7-CAT RNA was mostly uncapped but owing to the large amount it was translated, albeit
- RNA made by T7 RNA polymerase is not as efficiently capped as RNAs made by the vaccinia virus polymerase (Fuerst et al, supra) . Since the cap is needed for both RNA stability and translation [Grifo et al, 1982, in Interaction of Translational & Transcriptional Controls in the Regulation of Gene Expression eds. Grunberg-Manago, M. & Safer, B. , (New York: Elsvier Biomedical) , pp. 359- 372], this deficiency presented a severe cha.i ⁇ ⁇ > ⁇ .
- RNA stability problem was solved by retaining the T7 stem-loop segment at the 5' ends of the transcripts.
- This stem-loop nevertheless, may have exacerbated the capping problem since in vitro studies indicated that RNA containing this structure was a poorer substrate for the vaccinia RNA guanylyl transferase (Fuerst et al, supra) .
- the stem-loop structure might interfere with ribosome scanning (Pelletier et al, 1985, Cell 40, 515-526; Kozak, M. , 1980, Cell 19, 79-90).
- RNA generated approximately 30% of the total cytoplasmic RNA
- useful amounts of protein were made with the vaccinia/T7 hybrid expression system. It is important to point out here that at the start of the present study, the applicants did not know whether the EMCV UTR could function in a eukaryotic cell when attached to an RNA made by a prokaryotic transcription system. Further more, since vaccinia virus inhibits host cell protein synthesis, it was a matter of concern that the changes in the protein synthesis machinery of the cell caused by vaccinia virus might prevent the efficient use of the EMCV UTR.
- the mammalian cell expression system of the present invention is truly eclectic in that the vector is a DNA virus, the RNA polv_ ⁇ iP ⁇ - « ⁇ e --nri promoter are derived from a DNA bacteriophage, and the ribosome binding site is from an RNA virus. Nevertheless, the system provides a powerful, rapid, versatile and simple way of expressing genes in mammalian or avian cells.
- results with the small 25,000 dalton CAT protein revealed that it was the dominant polypeptide made in vaccinia virus-infected cells and after only 24 hr accumulated to a level of 16.5 ⁇ g per 2xl0 6 cells which accounted for approximately 10% of the Coomassie brilliant blue stained protein on polyacrylamide gels.
- the level of expression with the vaccinia/T7/EMCV hybrid system is greater than that obtained with conventional vaccinia virus vectors even when the strongest poxvirus promoters described thus far are used (Falkner, et al, 1988, J. Virol. 62, 1849-1854; Patel et al, 1988, Proc. Natl. Acad. Sci. USA 85, 9431-9435).
- the vaccinia hybrid system has other advantages, such as use for potentially toxic genes, since expression does not occur until cells are co-infected with the T7 RNA polymerase containing helper virus. Thus, the expression of toxic genes to kill target cells can be controlled by this unique feature of the present invention.
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Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US07/376,687 US5135855A (en) | 1986-09-03 | 1989-07-07 | Rapid, versatile and simple system for expressing genes in eukaryotic cells |
US376687 | 1989-07-07 | ||
PCT/US1990/003687 WO1991000905A1 (en) | 1989-07-07 | 1990-07-03 | Rapid, versatile and simple system for expressing genes in eukaryotic cells |
Publications (3)
Publication Number | Publication Date |
---|---|
EP0484374A1 true EP0484374A1 (en) | 1992-05-13 |
EP0484374A4 EP0484374A4 (en) | 1993-05-05 |
EP0484374B1 EP0484374B1 (en) | 1997-09-03 |
Family
ID=23486043
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP90910982A Expired - Lifetime EP0484374B1 (en) | 1989-07-07 | 1990-07-03 | Rapid, versatile and simple system for expressing genes in eukaryotic cells |
Country Status (9)
Country | Link |
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US (1) | US5135855A (en) |
EP (1) | EP0484374B1 (en) |
JP (1) | JP2525289B2 (en) |
AT (1) | ATE157701T1 (en) |
AU (1) | AU642657B2 (en) |
CA (1) | CA2063427C (en) |
DE (1) | DE69031389T2 (en) |
ES (1) | ES2109924T3 (en) |
WO (1) | WO1991000905A1 (en) |
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EP0628083A1 (en) | 1992-02-27 | 1994-12-14 | Microbial Technics Limited | Heterologous gene expression in lactococcus, and the expression products therefrom |
IL102404A0 (en) * | 1992-07-03 | 1993-01-14 | Daniel Zurr | Method and means for the production of gene products |
US5648235A (en) * | 1992-07-03 | 1997-07-15 | Q.B.I. Enterprises Ltd. | Method and means for the production of gene products, novel recombinant DNA vectors therefor and kits employing them |
US5409823A (en) * | 1992-09-24 | 1995-04-25 | Ciba-Geigy Corporation | Methods for the production of hybrid seed |
WO1994023745A1 (en) * | 1993-04-09 | 1994-10-27 | The University Of Virginia Patents Foundation | 35/31 kda subunit of the entamoeba histolytica adherence lectin |
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GB8613481D0 (en) * | 1986-06-04 | 1986-07-09 | Diatech Ltd | Translation of mrna |
US4937190A (en) * | 1987-10-15 | 1990-06-26 | Wisconsin Alumni Research Foundation | Translation enhancer |
IL91935A0 (en) * | 1988-10-11 | 1990-06-10 | Us Commerce | New plasmid constructions for high level production of eukaryotic proteins |
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1989
- 1989-07-07 US US07/376,687 patent/US5135855A/en not_active Expired - Lifetime
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1990
- 1990-07-03 EP EP90910982A patent/EP0484374B1/en not_active Expired - Lifetime
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- 1990-07-03 JP JP2510227A patent/JP2525289B2/en not_active Expired - Lifetime
Non-Patent Citations (4)
Title |
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JOURNAL OF MOLECULAR BIOLOGY vol. 206, no. 2, 20 March 1989, pages 333 - 348 FUERST, T. & MOSS, B. 'Structure and stability of mRNA synthesized by Vaccinia Virus-encoded bacteriophage T7 RNA polymerase in mammalian cells . Importance of the 5' untranslated leader' * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 86, no. 16, August 1989, WASHINGTON US pages 6126 - 6130 ELROY-STEIN, O. ET AL. 'Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system' * |
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AU642657B2 (en) | 1993-10-28 |
ATE157701T1 (en) | 1997-09-15 |
ES2109924T3 (en) | 1998-02-01 |
AU6034490A (en) | 1991-02-06 |
CA2063427C (en) | 1999-09-21 |
US5135855A (en) | 1992-08-04 |
EP0484374B1 (en) | 1997-09-03 |
EP0484374A4 (en) | 1993-05-05 |
WO1991000905A1 (en) | 1991-01-24 |
DE69031389D1 (en) | 1997-10-09 |
DE69031389T2 (en) | 1998-03-19 |
CA2063427A1 (en) | 1991-01-08 |
JP2525289B2 (en) | 1996-08-14 |
JPH05501048A (en) | 1993-03-04 |
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